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Effect of estrogen on gene expression in the chick oviduct. Effect of estrogen on the sequence and population complexity of chick oviduct poly(A)-containing RNA.

Total cellular RNA preparations were isolated from chicken oviducts at three different development stages: (a) immature chicks which were chronically stimulated with estrogen; (b) estrogen-stimulated chicks which were then withdrawn from hormone for 12 days; and (c) laying hens. Total cellular RNA containing 3'-poly(A) sequences (poly(A)-RNA) were than isolated from these preparations using oligo(dT)-cellulose chromatography. The number average nucleotide length of the poly(A)-RNA preparations in each case was approximately 2000 nucleotides. The number average nucleotide length of the poly(A) residues at the 3'-terminal end of each RNA preparation was approximately 70 adenylate residues. Complementary DNA (cDNA) copies to each preparation of poly(A)-RNA were synthesized using avian myeloblastosis virus RNA-directed DNA polymerase. The cDNApoly(A) preparations were then utilized in DNA excess hybridization experiments to analyze the complexity of the DNA sequences from which these RNAs were transcribed. Approximately 22% of each of the total cellular poly(A)-RNAs were transcribed from repeated DNA sequences (average repeat frequency of 35 copies/genome) while the remaining majority were transcribed from single copy or unique sequence DNA. It was possible to estimate the number of different poly(A)-RNA sequences per cell by analyzing the kinetics of hybridization of these cDNApoly(A) preparations to total cellular poly(A)-RNA extracts under conditions of RNA excess. The results revealed that 41% of the poly(A)-RNA from laying hen oviduct consisted of, on the average, three different sequences/cell, each of which was present in approximately 25,000 copies/cell. The remainder of the poly(A)-RNA in this tissue consisted of approximately 25,000 different sequences/cell, which were present largely in only two or three copies/cell. A somewhat similar sequence complexity was found for oviduct cells prepared from estrogen-stimulated chicks. We estimated that there were approximately 20,000 different poly(A)-RNA sequences/cell, each represented in only one to two copies/cell. However, there were five sequences which were present, on the average, in a concentration of 5600 copies/cell. The poly(A)-RNAs from hormone-wtihdrawn tissue, on the other hand, had a lower sequence complexity. There were only approximately 10,000 different poly(A)-RNA sequences/cell, each present in about three copies/cell. Furthermore, the few sequences present in a great abundance in hen and hormone-stimulated tissues were apparently absent in oviduct tissue from hormone-wtihdrawn chicks, suggesting that the intracellular concentrations of these high frequency RNA sequences are dependent on estrogen.

Animals↗

Localization of macrophages in the chicken oviduct: effects of age and gonadal steroids.

The aim of this study was to localize macrophages in the hen oviduct and determine the effects of age and gonadal steroids on their population. Cryostat sections of oviducal tissues from immature hens (60 d of age), virgin young (175 d of age), and old (620 d of age) laying hens, and immature hens (84 d of age) treated with gonadal steroids were immunostained for macrophages. The population of macrophages was analyzed by an image analysis system under a light microscope. Macrophages were observed in the stroma and mucosal epithelium of all oviducal segments of immature and laying hens. The population of macrophages in the oviducal stroma increased with age. Young laying hens had a significantly higher population of macrophages than immature hens in the vagina. Old laying hens had a significantly higher population than immature hens in the infundibulum, magnum, and vagina, and than young laying hens in the magnum. In the immature hens treated with gonadal steroids, the macrophage population increased only in the stroma of shell gland of the progesterone-treated birds. These results indicate that macrophage population in the oviducal stroma increases in association with sexual maturation and aging. Their population in the stroma may be partially affected by progesterone.

Aging↗

Cyclic nucleotides and prostaglandins (PGs) produced by the rabbit oviduct: effects of estrogen treatment.

Levels of cyclic AMP and cyclic GMP have been measured in oviducts of rabbits killed 68 hr after injection of human chorionic gonadotrophin (HCG) to induce ovulation. cAMP and cGMP levels were higher in the isthmus than in the ampulla. Estrogen treatment given at the same time as HCG increased cGMP levels. Levels of PGE and PGF in blood draining the oviduct were low and showed no effect of estrogen treatment at 68 hr after HCG.

Animals↗

Adrenergic influences on rabbit oviduct: effect of muscle size and ovarian hormones.

This study attempts to distinguish between a direct action of ovarian steroids on adrenergic neurons in the oviduct and an indirect effect mediated by changes in muscle size. Mature rabbits were treated as follows: group 1, ovariectomized and a polyethylene catheter (1 mm OD) inserted into isthmus of one oviduct (CT) with contralateral oviduct as control (C); group 2, normal, estrous animals with one intubated isthmus (ET) and contralateral control (E). Fourteen days postoperatively, oviducts were removed and muscle-wall thickness, norepinephrine (NE) content, and response to nerve stimulation were measured. Although castration atrophy was prevented in CT, NE content of CT was significantly less than C, 0.032 +/- 0.07 versus 1.09 +/- 0.10 nmol. NE content of ET was also significantly less than E (1.32 +/- 0.03 versus 1.81 +/- 2.0) despite a greater wall thickness of ET. It was concluded that: a) withdrawal of ovarian hormones reduces NE contents by a direct action on nerves; b) moderate stretch, per se, increases muscle size and reduces NE content; c) nerve stimulation induces muscle contraction despite large reduction in transmitter content.

Adrenergic Fibers↗

Nuclear synthesis of egg white protein messenger ribonucleic acids in chick oviduct: effects of the anti-estrogen tamoxifen on estrogen-, progesterone-, and dexamethasone-induced synthesis.

Tamoxifen is a potent anti-estrogen in the chicken oviduct [Sutherland, R., Mester, J., & Baulieu, E.E. (1977) Nature (London) 267, 434-435]. Its action on egg white protein gene transcription was studied in isolated nuclei under various hormonal conditions. Injected alone to estrogen-primed and then withdrawn chickens, tamoxifen was unable to trigger gene transcription. After its administration together with or 6 h after diethylstilbestrol (a synthetic estrogen), tamoxifen stopped or suppressed the estrogen-dependent increase of ovalbumin and conalbumin gene transcription. On the contrary, when tamoxifen was given with progesterone or with dexamethasone (a synthetic glucocorticosteroid), two steroids that also increased specific transcription of the ovalbumin and conalbumin genes, there was an amplification effect that lasted up to 24-30 h. These results demonstrate that tamoxifen is active at the transcriptional level when inhibiting estrogen action and when increasing progesterone and dexamethasone effects on protein synthesis [Catelli, M. G., Binart, N., Elkik, F., & Baulieu, E. E. (1980) Eur. J. Biochem. 107, 165-172; Le Bouc, Y. (1983) Thèse de 3ème cycle, Université Paris VII]. The complexity of hormone-anti-estrogen interactions on transcriptional efficiency was also illustrated by the greater amplifying effect of tamoxifen on conalbumin than on ovalbumin gene transcription and by the lack of potentiation by the anti-estrogen of dexamethasone-dependent ovomucoid gene transcription. The role of tamoxifen-estrogen receptor complexes in these responses is discussed in view of their differential amount in chromatin in the presence of estrogen or of progesterone.

Animals↗

Quantitative estimates of cytoplasmic and nuclear oestrogen receptors in chick oviduct. Effect of oestrogen on receptor concentration and subcellular distribution.

(3H)Oestradiol exchange techniques were developed for the determination of specific oestrogen receptor site concentrations in the cytoplasm and nuclei of chick oviduct cells. Non-labelled, receptor-bound oestrogens were exchanged with (3H)oestradiol during a 24-h incubation at 20 degrees C, 2 h at 30 degrees C or 45 min at 3 degrees C. Both "soluble" and "insoluble" nuclear receptors were stable for at least 6 h at 30 degrees C and 3 degrees C but a proportion (approx. 30%) of cytoplasmic sites from withdrawn chickens were inactivated after 2 h at 20 degrees C. The magnum of 4-week-old immature chickens (weight = 15 mg) contained 0.20 pmol of oestrogen receptor which corresponds to 4275 receptor sites/cell, when it is assumed that all magnum cells have equal concentrations of receptor. In primarily stimulated chickens of similar age which had received 10x1 mg of oestradiol benzoate/day, the magnum weighed approximately 800 mg and contained 8.65 pmol of oestrogen receptor (4610 sites/cell). Withdrawal from primary oestrogenic stimulation for 3-6 weeks resulted in a 110 mg magnum which contained 1.20 pmol of receptor (2225 sites/cell). Oviducts from immature and withdrawn chickens had the majority (73-77%) of their oestrogen receptors sites in the receptor sites in the cytoplasmic fraction, while in primary stimulated chicken oviducts the majority (82%) of receptor sites were located in the nucleus. A single secondary injection of oestradiol, to oestrogen-withdrawn chickens, resulted in apparent translocation of cytoplasmic receptors to the nucleus during the first hour after injection. The magnitude of the decline in cytoplasmic receptor, and the concurrent increase in nuclear receptor concentration, was dose-dependent between 2 and 100 mug oestradiol/kg body weight. Larger doses of oestradiol up to 1 mg/kg did not increase the concentration of nuclear receptor above the maximum level seen at 100 mug oestradiol/kg. The initial rapid accumulation of nuclear receptor sites was followed by a period of progressive decline. The initial rapid accumulation of nuclear receptor sites was followed by a period of progressive decline. By 15 h after an injection of 100 mug oestradiol/kg, the concentration of nuclear sites had reached pre-injection levels. During the same time period, the depleted cytoplasmic receptor levels were replenished such that they reached control values by 12 h and were about 150% of the pre-injection level at 24 h.

Animals↗

Effects of prostaglandins on avidin induction in the chick oviduct.

Effects of exogenous prostaglandins (PGs) on avidin synthesis in the oviduct were studied in immature diethylstilboestrol-treated chicks. Among eight tested PGs only PGF-2alpha was effective when injected directly into the oviduct, but intraperitoneal and intravenous administration also caused avidin induction. 1 ng of PGF-2alpha/100 g B.W. injected into the oviduct caused avidin synthesis. The effect of PGF-2alpha on avidin induction occurred as early as 3 hr after intraoviductal administration. This study provides an additional evidence for the hypothesis that PGF-2alpha may be involved in the mechanism of avidin induction in the chick oviduct.

Animals↗

Effect of estrogen on angiotensin converting enzyme in immature quail oviduct.

Effect of oestradiol was studied on the angiotensin converting enzyme (ACE)--a component of renin angiotensin system, in oviduct of immature quails of 15 days of age. ACE was studied in whole oviduct, magnum, shell gland and the glandular epithelium of magnum and shell gland. It was found that whole oviduct had a significantly higher level of ACE in control than those treated with exogenous estrogen at three dose levels (200, 400 or 600 micrograms). ACE contents of whole muscle and glandular epithelium did not differ but magnum had higher ACE level than the shell gland. Results are explained on the basis of functional role of oviductal parts.

Analysis of Variance↗

The change in oestrogen and progesterone levels triggers adovarian propulsive movement of the hamster oviduct.

Effects of oestrogen and progesterone on oviduct movement were observed in vivo in ovariectomized hamsters. Continuous administration of oestradiol via implanted Silastic tubing did not induce co-ordinated propulsive movement of the oviduct towards the ovary, but the withdrawal of the hormone did. The most efficient adovarian propulsion was observed when the withdrawal of oestradiol was combined with progesterone administration. Progesterone alone did not stimulate this type of movement.

Animals↗

Coitus induced changes in oviductal motility & effect of progesterone.

Motility of different oviductal segments of conscious rabbits was recorded through permanently implanted sensors using the technique of impedance plethysmography. The implants were around the oviductal wall and therefore did not obstruct its lumen. Pre-ovulatory ampullary motility was always less than the isthmic motility. Coitus induced ovulation produced a characteristic oviductal motility pattern consisting of (i) initial relaxation of both isthmus and ampulla (4-12 h) followed by (ii) increased isthmic motility in the face of a continually relaxed ampulla (36-48 h), and finally phase (iii) leading to restitution of both ampullary and isthmic motility to the base-line at 72-96 h. Estimation of ova positions indicated the presence of fertilized eggs in the ampulla and ampullo-isthmic junction at 48 h and the ova could come to the end of the isthmic segment only at 72 h or after. Increased isthmic motility thus served to counter the transportation of ova and their retention in the ampulla. Rabbits in which oviducts were not taken out for ova positioning achieved normal pregnancy. Administration of progesterone (im, 2.5 mg) produced complete relaxation of both isthmus and ampulla, did not produce increased isthmic contractility on coitus, accelerated the ovum transport rate and inhibited pregnancy, again emphasising the ova retentive role of oviductal motility.

Animals↗

Effects of oviductal fluid on sperm penetration and cortical granule exocytosis during fertilization of pig oocytes in vitro.

The effects of oviductal fluid on sperm penetration and cortical granule exocytosis in pigs were examined. Cortical granule exocytosis in oocytes matured in vivo and in vitro was observed by staining with fluorescent-labelled lectin and laser-scanning confocal microscopy. Exocytosis of matured oocytes was classified into three categories after in vitro fertilization: complete cortical granule exocytosis and even distribution of exudate in the entire perivitelline space (type I); complete exocytosis and partial distribution of exudate (type II) and incomplete cortical granule exocytosis (type III). The incidence of oocytes with type I exocytosis was higher in oocytes matured in vivo than in those matured in vitro. The addition of oviductal fluid at a concentration of 1% or 10% to the fertilization medium decreased sperm penetration and the mean number of spermatozoa present in penetrated eggs. The distribution of cortical granule exudate was not different in the presence of 1% oviductal fluid after sperm penetration from that of control groups. When oocytes were cultured for 1.5 h in medium containing 10% or 30% oviductal fluid before insemination, the incidence of monospermy increased without a decrease in sperm penetration. Preculture of oocytes in medium containing 30% oviductal fluid increased type I cortical granule reaction and increased resistance of the zona pellucida to dissolution by 0.1% (w/v) pronase at the time of sperm penetration. These results suggest that a factor(s) from the oviductal secretion is required for the complete cortical granule reaction and in the modification of the zona pellucida.

Acrosome↗

Spike bursts in rabbit oviduct. I. Effect of ovulation.

Electrical activity of rabbit oviducts was recorded in vitro at 18, 24, and 68 h after injection of human chorionic gonadotropin. An array of eight suction electrodes 1-2 mm apart was moved sequentially to cover nearly the whole oviduct. The frequency of spike bursts, pacemaker frequency, percentage of nonpropagating bursts, and probability of propagating in the uterine direction varied steeply within a few millimeters along the oviduct. On the average, spreading was only slightly prouterine. Compared to 18 and 24-h, 68-h oviducts showed higher frequency of activity and longer distances of propagation, particularly in the isthmus. The activity extended to 65.8 and 69.6% of the distance from the fimbriae at 18 and 24 h, respectively, and at 68 h to 70.8% and 84.6% in those oviducts that contained and did not contain ova, respectively. Ova were recovered on the uterine side of the border of activity or in regions in which the percentage of nonpropagating bursts was high or, at 18 and 24 h, in segments containing local regions of inactivity. Apparently, frequency of activity, the degree to which propagation of activity deviates from randomness, and the length of the inactive area regulate the location of ova in the isthmus.

Action Potentials↗

Effects of oviductal fluid and heparin on fertility and characteristics of porcine spermatozoa.

The objective of this study was to determine the effects of oviductal fluid and heparin on sperm penetration and the characteristics of spermatozoa. The addition of oviductal fluid and heparin to the fertilisation medium decreased sperm penetration and the mean number of spermatozoa in penetrated eggs. The number of spermatozoa firmly bound to zona pellucida was also decreased in the presence of oviductal fluid and heparin. Chlortetracycline (CTC) fluorescence patterns were used to determine the incidence of capacitation and the acrosome reaction. The proportion of capacitated and acrosome-free spermatozoa increased when spermatozoa were exposed for 1.5 and 3 h to oviductal fluid and heparin. In contrast heparin alone did not increase the number of capacitated spermatozoa at these time points. These results suggest that factor(s) in oviductal secretions reduce polyspermic fertilisation and the number of spermatozoa that will penetrate porcine oocytes. The reduction of polyspermic penetration by oviductal secretions may be due to a reduced number of spermatozoa in the fertilisation medium with an intact acrosome.

Animals↗

Electrical activity of the smooth muscle of guinea-pig oviduct and effects of catecholamines.

The membrane activity of the oviductal smooth muscle cell of the nonpregnant guinea pig was studied intracellularly. The membrane potential was about 50 mV. The cells used were spontaneously active in normal solution. Spontaneous action potentials were recorded from the regions of infundibulum, isthmus, and ampulla of the oviduct. Action potentials were composed of repetitive spikes and a plateau phase and the duration of the plateau phase ranged from 2 sec to 6 sec. The amplitude of the plateau phase was about 20 mV. The shape of the spontaneous action potential obtained from the mesosalpinx differed from that of the oviduct. Adrenaline, noradrenaline, and phenylephrine evoked the excitatory action. In general, small depolarization, increase in spikes on a plateau, and prolongation of the plateau duration occurred because of these drugs. After the treatment with phentolamine, adrenaline inhibited spike generation. Isoprenaline decreased the number of spikes on the plateau and the plateau duration. At higher concentration of isoprenaline, the membrane activity was abolished. The inhibitory effect of isoprenaline was blocked by propranolol. Prostaglandin F2-alpha excited membrane activity. These results indicate that the sympathetic innervation of the oviductal smooth muscle is alpha-excitatory and beta-inhibitory.

Action Potentials↗

Effect of oviductal epithelial cells on fertilization of pig oocytes in vitro.

The incidence of polyspermy is reduced by co-culture of pig oocytes with oviductal cells. It is not known whether the effect is due to soluble factors secreted into the medium. Oviductal epithelial cell monolayers and cell-conditioned media were prepared and their effects on fertilization of pig oocytes were examined. In vitro matured pig oocytes were inseminated with ejaculated boar spermatozoa at a concentration of 1x10(5) or 1x10(6) cells/ml and co-cultured in one of 5 culture systems: an oviductal epithelial cell monolayer, a fibroblast monolayer, an oviductal epithelial cell-conditioned medium, or a fibroblast-conditioned medium, and medium alone (modified-TCM199). In all 5 systems, the majority (range 85 to 100%) of the oocytes were penetrated by sperm. When oocytes were inseminated with spermatozoa at a concentration of 1x10(5) cells/ml, the percentages of monospermic oocytes were significantly higher in the oocytes co-cultured with oviductal epithelial cells and fibroblasts than that of the oocytes cultured without these cells. In contrast, when oocytes were inseminated with spermatozoa at a concentration of 1x10(6) cells/ml, the percentages of monospermic oocytes were significantly higher in the oocytes co-cultured with epithelial cells than those cultured with the fibroblasts and in the control medium. The suppressive effect on polyspermy was observed in the oviductal epithelial cells-conditioned medium when oocytes were inseminated with spermatozoa at both concentrations of 1x10(5) and 1x10(6) cells/ml. The effect was absent in the fibroblasts-conditioned medium. Moreover, the effect of the epithelial cells was maintained during the culture period, whereas the proportion of monospermic oocytes co-cultured with fibroblasts showed a gradual decrease, reaching 0% after 16 h. These results suggest that a soluble factor(s) derived from the oviductal epithelial cells decreased the number of spermatozoa penetrating the oocytes without suppressing the high rate of fertilization.

Journal Article↗

Effect of oviductal proteins on sperm functions and lipid peroxidation levels during cryopreservation in buffaloes.

A study was undertaken to find out the effect of addition of oviductal proteins on sperm functions and lipid peroxidation (LPO) levels in buffaloes. Oviductal flushings were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle), centrifuged (3000 rpm; 30 min), filtered (0.2 microm) and frozen at -20 degrees C. The proteins in pooled nonluteal and luteal oviductal fluid were precipitated overnight using ammonium sulphate, centrifuged (10,000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored frozen at -20 degrees C. Six pooled good quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was split into three parts and extended in Tris-Egg yolk-Citrate extender (20% egg yolk: 7% glycerol), so that final dilution yielded approximately 60 million sperm cells/ml and cryopreserved in 0.5 ml French straws (30 million sperm cells per straw) in LN2 (-196 degrees C). Before freezing, the nonluteal and luteal oviductal proteins (NLOP &LOP) were incorporated at the concentration of 1mg/ml of extended semen. The equilibrated and frozen thawed (37 degrees C for 30s) semen was evaluated for motility, viability and acrosomal integrity, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides these tests, LPO level was assessed in sperm and seminal plasma in equilibrated and frozen thawed semen. Results revealed that addition of oviductal proteins to semen before freezing convey beneficial effect in terms of spermatozoan motility, viability and acrosomal integrity. Nonluteal oviductal proteins favored significantly (P < 0.05) higher sperm penetration distance in cervical mucus (23.00+/-1.15 mm) than the control group (15.00+/-3.46 mm) in frozen thawed semen. Similarly, swollen sperm percentage was also significantly (P < 0.05) higher in NLOP treated group than the LOP included and control groups. In frozen thawed spermatozoa, the LPO level was significantly (P < 0.05) lower in NLOP added group than the LOP added and control group. It was inferred that incorporation of oviductal proteins in extender before freezing reduced the lipid peroxidation levels in buffalo spermatozoa during cryopreservation and thereby improved the post-thaw semen quality.

Acrosome↗